The Confocal Imaging Facility (CIF) of the NIA IRP has been in operation since October 2004. This facility started with one older model (Zeiss LSM 410) confocal system and has successfully integrated the Zeiss LSM 510 Meta confocal system in 2005 and the Zeiss 710 confocal system in 2010. We expect to add a super-resolution system in FY 2016. In addition, we have updated the Zeiss Axioskop fluorescent microscope to a dual use system: MCID/QImage analysis system and Metamorph/Coolsnap imaging and analysis system. This expansion and upgrading reflects the extensive use of confocal resources by intramural scientists at different levels of expertise, ranging from principal senior investigators through postdoctoral fellows and intramural research trainees. In FY05 there were 15 users from 5 Labs, while by FY2009 the CIF was used by over 60 researchers from 10 Labs, including 45 investigators trained by the CIF staff to be independent users of the confocal microscopes. These trends have continued, and by FY15 approximately 90 different investigators have used the CIF and over 70 were trained by us. In addition to confocal usage, the MCID imaging system was being used by 8 other researchers. As a resource for all matters pertaining to microscopy and imaging, we have held scores of consultations with NIA Lab Chiefs, PIs and both intramural and extramural researchers to assist with their imaging and image analysis. We have helped users integrate imaging into their experimental systems, perform immunofluorescence studies, and troubleshoot problems encountered by investigators in their projects. The following imaging techniques have been used or introduced at the CIF: (a) Precise sub-cellular localization and co-localization of proteins; (b) Investigation of intracellular and intranuclear protein trafficking; (c) Implementation of novel methodology to cause DNA damage to live cell DNA at the sub-micron level by continuous UV laser scanning; (d) Time lapse, FRAP and ratio-metric analysis of cellular processes in live cells; (e) Volumetric (3D) reconstruction of intracellular protein distribution using confocal or deconvolution techniques, and of organelles such as human pancreatic islets of Langerhans from optical Z-sections. (f) Devising novel, confocal-based methodology to investigate receptor activation using fluorescent ligands. (g) Evaluation of super-resolution microscope platforms as a research tool for introduction into the NIA IRP. The importance of these techniques is emphasized in our recent research. We have followed the intracellular trafficking of several proteins, together with the precise sub-cellular localization and high-resolution co-localization of these proteins in multi-component protein complexes. Thus, in collaboration with the lab of Dr. Weeraratna (NIA and Wistar Institute), these methods have yielded very important characterizations of invasive melanoma cells: (a) defining Wnt5a (a protein that increases melanoma metastasis) interaction with syndecan which is via sugar chains, and was not amenable to immunonoprecipitation analysis, but which were proven to bind using immunofluorescence and confocal microscopy (See publications 2006-2011, also recently (O'Connell et al., Cancer Discov. 3(12):1378-93, 2013); (b) Using primarily immunofluorescence and confocal microscopy, we show that phosphorylation modifications of the tight junction protein claudin-1 cause its translocation to the cytoplasm and nucleus and that the sub-cellular localization of claudin-1 may dictate the metastatic capacity of melanoma cells. Our findings suggest that nuclear versus cytoplasmic expression of claudin-1 may become a valuable marker for diagnosis of malignant melanoma (French et al., Int. J. Med. Sci. 6:93-101, 2009); (c) Trafficking of EGFR-GFP showed endocytosis to late endosomes and lysosomes in cells expressing filamin A (an actin-binding protein), but not in cells that do not express filamin A, suggesting that filamin A contributes to activation and sorting of EGFR, an important member of the receptor tyrosine kinase family, that is implicated in oncogenesis (Fiori et al., Endocrinology 150:2551-60, 2009). Another important protein complex was discovered in a different system. In research done in collaboration with the lab of Dr. Biragyn (LI), we used in a T cell system a very nice three-color co-localization technique to provide quantitative evidence for the interaction of CD45 (a phosphatase that regulates Lck), Lck (a Src kinase) and GCR (glucocorticoid receptor) in a cell membrane protein complex that is important for the activation of T cells (Baatar et al., Brain Behav. Immunol. 23:1028-37, 2009). In intranuclear research we have assisted in defining the interactions of RNA binding proteins (Indig et al., PLoS One, 7:e35229, 2012) with our collaborators at the University of Maryland. CIF staff (Sarah Subaran, B.Sc.) contributed to a high-profile research paper published last year in the journal Nature Structural and Molecular Biology (Lee et al., 2010, Nat Struct Mol Biol 17:732-9). This research, performed as part of our ongoing collaboration with the lab of Dr. Gorospe (LCMB), involves imaging RNA binding proteins that form cytoplasmic structures called stress granules (Lee et al., 2012, PNAS 109:5750-5). These structures are defined exclusively by immunofluorescence, underlying the importance of imaging to the mission of the intramural research at the NIA. Currently, the CIF is involved in several project with various labs at the NIA: (1) The investigation of Cannabinoid receptor binding in live cells (with Dr. Wainer, LCI and Dr. Bernier, TGB). This investigation utilizes fluorescently tagged ligands in order to follow receptor binding in live cells. This research has recently been published (Paul et al., Biochem Pharmacol. 15;87(4):547-61, 2014 and Habicht et al., Anal Biochem. 484:154-61, 2015). (2) The investigation of specific mitochondrial micro-RNA expression using confocal techniques, together with the lab of Dr. Gorospe, LG. Our work on RNA binding proteins and lncRNA has been submitted for publication. (3) Protein trafficking in neurons with Dr. Yao, LNS, published recently (Petralia et al., Neuromolecular Med. 15(1): 49-60, 2013). (4) Muscle mitochondria function and distribution, part of the CRB/BLSA Sarcopenia project (with Dr. Ferrucci, TGB, and Dr. Hari Shroff, NIBIB), in which we will apply advanced imaging techniques, such as super-resolution microscopy, to investigate mitochondrial function in aging human muscle tissue. An additional advantage of this project is the careful examination of different super-resolution systems on human tissue samples in order to evaluate which of these new techniques and platforms would be the most beneficial for research done at the NIA.